Cell Line Authentication and Drug Treatment

Human CRC cell lines were obtained from the American Type Culture Collection (Manassas, VA). FBW7-KO HCT116 cells were obtained from Horizon Discovery (Cambridge, UK). PUMA-KO HCT116 cells were previously described (18 (link)). Cell lines were authenticated in 2015 by genotyping, western blotting of protein expression, and routine detection of mycoplasma contamination by PCR. All cell lines were maintained at 37°C and 5% CO₂ in McCoy’s 5A modified media (Invitrogen) supplemented with 10% defined FBS (HyClone), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For drug treatment, cells were plated in 12-well plates at 20–30% density 24 hr before treatment. Chemicals, including regorafenib, sorafenib, UCN-01, sunitinib, roscovitine, sulindac sulfide, etoposide, MG132, ABT-737, ABT-263, SB216736, TW37 (Selleck Chemicals), cycloheximide (CHX; Sigma), and UMI-77 (19 (link),20 (link)), were solubilized in DMSO and diluted to appropriate concentrations with the cell culture medium. TRAIL (XcessBio, San Diego, CA) was diluted with distilled water.